Functional characterisation of G protein-coupled receptors

Romez Uddin, John Simms, David Poyner

Research output: Contribution to journalArticle

Abstract

Characterisation of receptors can involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions. This review focuses on G protein-coupled receptors (GPCRs), examining techniques that can be applied to both receptors in membranes and after solubilisation. Radioligand binding remains a widely used technique, although there is increasing use of fluorescent ligands. These can be used in a variety of experimental designs, either directly monitoring ligand itself with techniques such as fluorescence polarisation or indirectly via resonance energy transfer (fluorescence/Forster resonance energy transfer, FRET and bioluminescence resonance energy transfer, BRET). Label free techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are also increasingly being used. For GPCRs, the main measure of receptor activation is to investigate the association of the G protein with the receptor. The chief assay measures the receptor-stimulated binding of GTP or a suitable analogue to the receptor. The direct association of the G protein with the receptor has been investigated via resonance energy techniques. These have also been used to measure ligand-induced conformational changes within the receptor; a variety of experimental techniques are available to incorporate suitable donors and acceptors within the receptor.
LanguageEnglish
JournalMethods
Early online date3 Mar 2018
DOIs
Publication statusE-pub ahead of print - 3 Mar 2018

Fingerprint

G-Protein-Coupled Receptors
Ligands
Fluorescence Resonance Energy Transfer
GTP-Binding Proteins
Chemical activation
Association reactions
Bioluminescence
Calorimetry
Fluorescence Polarization
Surface Plasmon Resonance
Energy Transfer
Surface plasmon resonance
Guanosine Triphosphate
Titration
Design of experiments
Energy transfer
Labels
Assays
Research Design
Fluorescence

Bibliographical note

© 2018, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/.

Funding: BBSRC [grant number BB/M007529/1].

Cite this

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abstract = "Characterisation of receptors can involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions. This review focuses on G protein-coupled receptors (GPCRs), examining techniques that can be applied to both receptors in membranes and after solubilisation. Radioligand binding remains a widely used technique, although there is increasing use of fluorescent ligands. These can be used in a variety of experimental designs, either directly monitoring ligand itself with techniques such as fluorescence polarisation or indirectly via resonance energy transfer (fluorescence/Forster resonance energy transfer, FRET and bioluminescence resonance energy transfer, BRET). Label free techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are also increasingly being used. For GPCRs, the main measure of receptor activation is to investigate the association of the G protein with the receptor. The chief assay measures the receptor-stimulated binding of GTP or a suitable analogue to the receptor. The direct association of the G protein with the receptor has been investigated via resonance energy techniques. These have also been used to measure ligand-induced conformational changes within the receptor; a variety of experimental techniques are available to incorporate suitable donors and acceptors within the receptor.",
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Functional characterisation of G protein-coupled receptors. / Uddin, Romez; Simms, John; Poyner, David.

In: Methods, 03.03.2018.

Research output: Contribution to journalArticle

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